Proto-oncogene PBF regulation of cell adhesion and motility

Background and Aims: The proto-oncogene pituitary tumor-transforming gene (PTTG)-binding factor (PBF/PTTG1IP) is upregulated in thyroid cancer and associated with tumour progression. PBF potently induces thyroid cancer cell motility via Src kinase phosphorylation. We have recently shown that PBF is also required for physiological mouse embryonic fibroblast (MEF) motility. Pbf-knockout (KO) MEFs have significantly reduced migration and invasion compared with wild-type (WT) MEFs. Phosphoproteomic and RNA-Seq analyses revealed that PBF upregulation in Nthy-ori 3-1 thyroid cells altered expression and phosphorylation of key adhesion proteins. We hypothesised that PBF physiologically regulates cell adhesion, and its oncogenic expression promotes thyroid cancer cell motility via altered adhesion. This study aimed to further elucidate the regulation of cell adhesion by PBF.

Methods: We utilised Pbf-KO MEFs and CRISPR/Cas9-mediated PBF-KO TPC-1 human papillary thyroid carcinoma cells in the analysis of cell adhesion and spreading on fibronectin-coated plates.

Results: Cell adhesion assays demonstrated that Pbf-KO MEFs exhibited markedly decreased cell-substrate adhesion compared with Pbf-WT MEFs. We then assessed focal adhesions (FAs), the large protein complexes that link the cell cytoskeleton to the extracellular matrix. Immunofluorescence staining of focal adhesion kinase (FAK), vinculin and paxillin revealed fewer and shorter FAs located predominantly around the periphery of Pbf-KO MEFs, in comparison with Pbf-WT MEFs, which displayed numerous, elongated FAs along actin fibres throughout the cells. TPC-1 PBF-KO cells also demonstrated decreased cell-substrate adhesion, as well as reduced cell spreading. In support of this, LifeAct-GFP live cell imaging suggested that both PBF-KO MEFs and TPC-1 cells had impaired cell spreading and loss of orientation.

Conclusions: Taken together, these findings provide new mechanistic insights into the regulatory role of PBF in thyroid cancer cell motility through cell adhesion dynamics. These findings also highlight potential avenues to therapeutically target PBF regulated pathways in tumorigenesis.